Methods of treating pulmonary sarcoidosis

ABSTRACT

Mucolytic agents for use in treating pulmonary sarcoidosis are described herein. Patients in need of treatment for pulmonary sarcoidosis are administered a therapeutically effective amount of a mucolytic agent such as DNase I, Mesna or DiMesna. In some embodiments, the DNase I is a recombinant human DNase I such as dornase alfa.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional PatentApplication Ser. No. 62/047,361, filed Sep. 8, 2014, the disclosures ofwhich are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention is directed to methods for treating pulmonarysarcoidosis comprising the administration of mucolytic agents. Incertain embodiments, the methods comprise administering atherapeutically effective amount of DNase I to a patient havingpulmonary sarcoidosis.

BACKGROUND OF THE INVENTION

DNase I is an endonuclease found in mammals and other eukaryotes thatcleaves phosphodiester linkages in DNA. It acts on single-stranded DNA,double-stranded DNA, and chromatin to reduce the size of DNA strands andyield 5′-phosphate-terminated polynucleotides with a free hydroxylgroup. Human DNase I and certain variants are disclosed in U.S. Pat.Nos. 5,279,823; 6,348,343; and 6,391,607.

DNase I has been used to treat cystic fibrosis. Cystic fibrosis is adisease caused by mutations in a specific cellular chloride channelregulator, the cystic fibrosis transmembrane conductance regulatorprotein (CFTR). It is the most common autosomal recessive disease inCaucasians. The mutations prevent normal passage of Cl⁻ ions through thechloride channel lumen of the airway epithelial cell membranes,resulting in a relative impermeability to chloride ions in theepithelial cells of the lungs and a depleted airway surface liquidvolume. As a result of impaired function of the CFTR protein, mucusviscosity is increased and the thickened, tenacious secretions block theairways in the lungs of cystic fibrosis patients. The large amounts ofviscous mucus blocking the airways in the lungs of cystic fibrosispatients causes a propensity for chronic infection, resulting ininflammation, progressive airway and parenchymal damage, bronchiectasis,pulmonary exacerbations, lung function decline and frequently prematuredeath. Even though improved treatment has increased survival, the medianpredicted lifespan is only 35 years and patients experience significantmorbidity and hospitalizations. Approximately 95% of cystic fibrosisdeaths are due to lung infection.

In 1993, the U.S. Food and Drug Administration (FDA) approved aformulation of recombinant human DNase I for the treatment of cysticfibrosis. It was the first treatment approved by the FDA for cysticfibrosis in 30 years. The approved product is marketed in the U.S. byGenentech, Inc. under the brand name PULMOZYME®. PULMOZYME® is believedto act by cleaving DNA in the thick mucus secretions that are a hallmarkof cystic fibrosis. This tends to liquefy the mucus, making it easierfor the body to clear the mucus from the airways, with consequentimprovement in airway function and lessened susceptibility to bacterialinfections.

The success of PULMOZYME® in treating cystic fibrosis prompted its studyin bronchiectasis, another lung disease where mucus buildup was thoughtto play a role. Unfortunately, a large clinical trial of bronchiectasispatients not only failed to demonstrate any benefit from PULMOZYME®, butsuggested that such treatment was potentially harmful (O'Donnell, etal., 1998, Chest 113:1329-1334).

Sarcoidosis is a disease involving granulomas (abnormal collections ofinflammatory cells), often present as nodules, which can form in variousorgans, including the skin, heart, liver, lungs, nervous system, andgastrointestinal tract. The granulomas are characterized by theaccumulation of neutrophils, monocytes, macrophages, and activated Tcells, as well as the production of elevated levels of inflammatorymediators such as tumor necrosis factor-α (TNF-α) interferon-γ, andinterleukin-2.

The cause of sarcoidosis is unknown, but there is speculation that it istriggered by an immune reaction to some infectious or environmentalantigen that continues after exposure to the antigen ceases. The lung isthe most commonly involved organ in over 90% of cases. Most patients donot exhibit symptoms and are unaware that they have sarcoidosis. Half ofall asymptomatic sarcoidosis patients are diagnosed after routine chestx-ray. The most common presenting symptoms are cough and dyspnea. Themost common diagnostic clinical signs are: i) dyspnea, ii) cough, iii)skin rash, iv) inflammation of the eyes, v) weight loss, vi) fatigue,vii) fever, and viii) night sweats. Due to the non-specific nature ofgranulomas, sarcoidosis is generally diagnosed by excluding otherdiseases such as malignancies and infections. The lung in sarcoidosistypically displays the characteristic bilateral hilar lymphadenopathy onchest x-ray. Reversible stages of sarcoidosis (Scadding RadiographicStages I and II), characterized by nodular reticular infiltrates and“ground-glass” appearance of lung parenchyma on chest x-ray, may notrequire treatment. Irreversible sarcoidosis (Stages III and IV),characterized by pulmonary cysts, diffuse parenchymal lung disease,honey comb lung structure (due to consolidation of alveoli) andbronchiectasis, has poor long-term prognosis and a high incidence ofpulmonary exacerbations/relapses. Computerized tomography (“CT”)findings for Stages III and IV include bronchiolar nodules(bronchovascular and subpleural), thickened interlobular septae,pulmonary architectural distortion and conglomerate masses. In theapproximately 70% of sarcoidosis patients that do not require medicalintervention, symptomatic treatment usually consists of non-steroidalanti-inflammatory drugs (NSAIDs) such as ibuprofen or aspirin; however,approximately one third of all patients develop a progressive form ofchronic sarcoidosis that requires treatment. The first-line therapy forall such chronic sarcoidosis patients is oral steroids (e.g., prednisoneor prednisolone) which have significant adverse side effects(susceptibility to infection, osteoporosis and rib fractures fromcoughing, diabetes, mental confusion, fluid retention, fatigue, etc.)and are not usable for chronic or long term therapy because of theseadverse drug responses. In some patients, corticosteroids slow orreverse the course of the disease, but many patients may becomerefractory to steroids or do not respond at all. Those patients mayexperience frequent and severe respiratory infections associated withepisodes of excessive coughing to dislodge and expel inspissatedsecretions and cell debris, including granulomas shed into the bronchiallumen. In corticosteroid non-responders with severe symptoms and notreatment options, other cytotoxic agents such as azathioprine,methotrexate, mycophenolic acid, and leflunomide may be tried. Of these,methotrexate is most widely used and is considered a first-linetreatment in neurosarcoidosis, often in conjunction withcorticosteroids. In general, the cytotoxic agents do not have benefitsthat outweigh their increased morbidity from cytotoxicity. The onlydefinitive treatment for end-stage disease is lung transplantation andsuch patients with pulmonary sarcoidosis represent approximately 30% ofall lung transplants conducted in the New York Presbyterian, ThoracicSurgery Lung Transplant Service (J.R. Sonnet, personal communication).

Some success in treating sarcoidosis with immunosuppressants has beenobserved. The rationale for such treatment is that the granulomasinvolved in sarcoidosis are caused by collections of immune systemcells, particularly airway neutrophils and circulating T-cells.Infliximab, a monoclonal antibody that antagonizes the action of TNF-α,has been used to treat pulmonary sarcoidosis in clinical trials, withsome success. Etanercept (another TNF-α antagonist), on the other hand,failed to demonstrate any significant efficacy in people with uvealsarcoidosis. The anti-TNF-α monoclonal antibody golimumab also failed toshow any benefit in persons with pulmonary sarcoidosis. Adalimumab (yetanother anti-TNF-α monoclonal antibody) induced a beneficial response inabout half of sarcoidosis patients. See Baughman, et al., 2013, EuropeanRespiratory Journal 41:1424-1438.

Individualized therapy is a new paradigm in modern medicine as ittransitions from “blockbuster” drugs to stratified personalizedmedicine. Unfortunately, this approach is not optimally supported byaverage results from large randomized clinical trials (RCTs). The n=1approach confers extremely powerful assessment tools to achievepersonalized medicine and, using this approach, the patient is the soleunit of observation in therapeutic assessment. The advantages accruedare several. First, in a single patient study, heterogeneity in designis tolerated as long as the single patient stratification arm results inobjective evidence favoring the intervention, whereas largepopulation-based RCTs require design uniformity to prevent confoundinggeneralizations. Second, patients in an n=1 trial draw immediate benefitfrom the trial based on the optimal presentation and refinement ofintervention strategies designed to benefit them objectively. This istotally dissimilar to a population-based RCT wherein an individualpatient in physical distress may have received a placebo for the entirestudy period.

Surprisingly, the more powerful n=1 approach has only been usedsparingly in general clinical and medical settings, even though it isborn out of a recognition that medical interventions that work for amajority of chronic disease conditions have too often proven fruitlessin RCTs (Jorgensen, 2008, Expert Rev. Mol. Diagn. 8(6):689-695;Jorgensen, 2009, Oncologist 14(5):557-558). There is a growingpresumption that the clinical practice of medicine should recognize andembrace the unique individual characteristics of patients with rarediseases, often needing very costly treatment options, and strive toindividualize patient care (Hu et al., 2005, Biotechniques 39(10Suppl):S1-S6; Langreth & Waldholz, 1999, Oncologist 4(5):426-427;Trusheim et al., 2007, Nat. Rev. Drug Discov. 6(4):287-293).

In the present era of new drug development, large sample parallel groupRCT's are often begun without detailed knowledge of the optimaltherapeutic dose, patient selection criteria, and initial estimates ofthe proportion of patients that are responders (important for samplesize determination), optimal outcomes on which subsequent trials shouldbe based, safety for long-term/lifetime treatment, etc. In addressingmany of these issues in the current environment, where detailed,individualized information is available for single patients, the USNational Institutes of Health (NIH) has both advocated and acknowledgedthe utility of n=1 studies of patient responses to highly individualizedtherapy in forward-looking “precision medicine” evidence-basedapproaches. Individualized therapy improves outcome assessment becausethe treatment regimen is tailored to the patient's diseasestratification. For example, the anticancer drug cetuximab (colorectalcancer) is ineffective if the KRAS protein in the tumor has a specificmutation (Van Cutsem et al., 2009, N. Engl. J. Med. 360(14):1408-1417)and the US FDA has relabeled the drug to require genetic profilingbefore use. Many other drugs have variations in effectiveness in certainpatient strata that led to FDA relabeling (e.g., warfarin,carbamazepine, clopidogrel) (Flockhart et al., 2009, Clin. Pharmacol.Ther. 86(1):109-113; Topol, 2010, Sci. Transl. Med. 2(44):44cm22).Furthermore, the FDA is actively developing streamlined reviewapproaches to companion diagnostic tests with treatments where n=1protocols facilitate the approval process (Hamburg & Collins, 2010, N.Engl. J. Med. 363(4):301-304).

The n=1 clinical trial approach is very cost effective, but requiresmuch more time commitment and supervision by medical professionals,coupled with suitably long observation intervals, coincident in theindex patient with the normal time course of disease progression(interspersed with periodic “cessation of treatment” intervals (or“washout periods”), to test for spontaneous remission of disease). Notonly is this targeted n=1 long-term study approach cost effective, butit has also resolved many confounding ambiguities of treatment thatwould be present in a population study, e.g., diet and lifestylechanges, progression/regression of disease, meaningful patient benefit,etc. The n=1 approach reflects the trend toward “personalized medicine”of the future and has a high degree of quasi-statistical precisionbecause the patient serves as his or her own control, increasingconfidence in the results of customized treatment. In fact, n=1 clinicalstudies could be deemed virtually essential for evaluation of highlytargeted therapies, many of which may not even be amenable to RCTsbecause the between variance for treatments would be large relative tothe relatively small sample size for an extremely rare disease. In suchcases, the n=1 approach epitomizes the appropriateness of clinical trialdesigns which minimize the time a patient is given a suboptimalintervention. Moreover, sequential designs with lengthy data collectionprocesses are especially useful for rare, unique diseases (Everitt &Pickler, 2004, Statistical Aspects of The Design of Clinical Trials.Imperial College Press; London, UK; Gerss & Kopcke, 2010, Adv. Exp. Med.Biol. 686:173-190; Meinert & Tonascia, 1986, Clinical Trials Design,Conduct, and Analysis Monographs in Epidemiology and Biostatistics. Vol.469. Oxford University Press; NY, USA).

The ultimate clinically important issue for novel interventions withutility is generalizability of results to subpopulations. It is herethat n=1 clinical trials excel, enabling stratification of patients intogroups more or less likely to benefit from a specific treatment forpopulation-level association studies (Barlow et al., Strategies forStudying Behavior for Change. 3. Vol. 393. Pearson/Allyn and Bacon; MA,USA; Guyatt et al., 1986, N. Engl. J. Med. 314(14):889-892). Individualvariations in response to treatment reflect population variations and ifstratifications are well described (Kraemer et al., 2002, Arch. Gen.Psychiatry 59(10):877-883; Kent & Hayward, 2007, JAMA 298(10):1209-1212;Scuffham et al., 2010, J. Gen. Intern. Med. 25(9):906-913), n=1 clinicaltrials objectively quantify this variability and provide informedguidance for treating individual patients using their own data. Theefficiency of n=1 clinical trials in identifying and minimizingsuboptimal treatments is far greater than standard care utilizing RCTs,both improving patient management and resulting in cost savings.

SUMMARY OF THE INVENTION

Described herein are methods of treating pulmonary sarcoidosiscomprising administering to a patient in need thereof a therapeuticallyeffective amount of a mucolytic agent. In certain embodiments, themucolytic agent is DNase I, e.g., recombinant human DNase I (rhDNase I).In certain embodiments, the rhDNase I is the rhDNase I that is theactive ingredient of PULMOZYME®.

In certain embodiments, the mucolytic agent is selected from the groupconsisting of: sodium 2-sulfanylethanesulfonate (mesna, marketed in theU.S. as UROMITEXAN®), disodium 2,2′disulfanediyldiethanesulfonate(dimesna), and combinations thereof.

In certain embodiments, the methods comprise administering DNase I andan additional mucolytic agent. In certain embodiments, the additionalmucolytic agent is selected from the group consisting of mesna, dimesna,N-acetylcysteine, and combinations thereof. In addition to beingmucolytic agents, mesna, dimesna, and N-acetylcysteine are free-radicalscavengers. Pulmonary cellular antioxidant defense is significantlyreduced with increased tissue levels of destructive free radicals in theinflammatory response to pulmonary sarcoidosis (Boots, et al., 2009,Resp. Med., 103:364). This finding points to the added utility ofconcomitant use of free-radical scavenging compounds such as mesna,dimesna and N-acetylcysteine. In those embodiments where a rhDNase Isuch as PULMOZYME is administered to the patient with an additionalmucolytic agent, the rhDNase I may be administered before, after, orconcomitantly with the additional mucolytic agent. Preferably, therhDNase I is administered before or after the additional mucolyticagent. In some embodiments, rhDNase I is administered by nebulizationand and mesna or dimesna is administered intravenously. In suchembodiments, mesna or dimesna act as free-radical scavengers.

In certain embodiments, the methods treat a patient who has acutepulmonary sarcoidosis (patient diagnosed with sarcoidosis for <2 years).In certain embodiments, the methods treat a patient who has chronicpulmonary sarcoidosis (patient diagnosed with sarcoidosis for ≧2 years).In certain embodiments, the methods safely treat a patient with chronicpulmonary sarcoidosis for periods in excess of 12 years.

Patients that progress to chronic pulmonary sarcoidosis deterioratefairly quickly due to recurrent infections/pulmonary fibrosis and don'tgenerally live for 12 years. Treatment with a rhDNase I such asPULMOZYME® could be long-term (over 12 years) without progression of, orwith minimal progression of, the disease or recurring pulmonaryexacerbations. It is possible that the progression to pulmonary fibrosisand death may be precluded by preventing the recurrent airway infectionswith PULMOZYME®, effectively preventing the post-obstructive pneumoniasand tissue damage by chemotactically attracted neutrophils that releaseneutrophilic proteases, inflammatory cytokines, nitric oxide, and oxygenfree radicals (which collectively are probably responsible for theobserved pulmonary fibrosis and emphysema-like loss of pulmonary realestate).

A key question relating to use of a rhDNAse I such as PULMOZYME® inpulmonary sarcoidosis patients is: “Is long-term use tolerated by thepatient and does the clinical condition deteriorate as with untreated orsteroid refractory disease?” The experimental results described herein—apatient who has undergone over 12 years of therapy with PULMOZYME®without side effects or incident, completely absent of any pulmonaryexacerbations—indicate that the answer to this question is “Yes.”

PULMOZYME® breaks the cycle of bronchial damage, impaired mucusclearance, recurrent inflammation and more damage—leading to pulmonaryfibrosis and/or hemoptysis and death in the most seriously afflictedpulmonary sarcoid patients.

The methods described herein may improve the health of pulmonarysarcoidosis patients by decreasing the need for other medications (e.g.,corticosteroids), reducing coughing, decreasing the number and severityof bacterial infections, improving the oxygen saturation of thepatient's blood, and/or allowing for greater physical activity (e.g.,improving exercise tolerance).

Certain embodiments of the invention include:

1. A method of treating pulmonary sarcoidosis comprising administeringto a patient in need thereof a therapeutically effective amount of amucolytic agent. Other embodiments include:

2. The method of embodiment 1 where the mucolytic agent is DNase I.

3. The method of embodiment 1 where the mucolytic agent is selected fromthe group consisting of: sodium 2-sulfanylethanesulfonate, disodium2,2′-disulfanediyldiethanesulfonate, and combinations thereof.

4. The method of embodiment 1 where the DNase I is recombinant DNase I.

5. The method of embodiment 4 where the recombinant DNase I isrecombinant human DNase I.

6. The method of embodiment 5 where the recombinant human DNase I isadministering to the patient's lungs by inhalation.

7. The method of embodiment 6 where the inhalation carried out with theuse of a nebulizer.

8. The method of embodiment 5 where the recombinant human DNase I hasthe amino acid sequence of native human DNase I.

9. The method of embodiment 5 where the recombinant human DNase I hasthe amino acid sequence of SEQ ID NO. 3.

10. The method of embodiment 1 where the pulmonary sarcoidosis is acutepulmonary sarcoidosis.

11. The method of embodiment 1 where the pulmonary sarcoidosis ischronic pulmonary sarcoidosis.

12. The method of embodiment 1 comprising administering an antibiotic tothe patient.

13. The method of embodiment 1 comprising administering a bronchodilatorto the patient.

14. The method of embodiment 1 comprising administering chest physicaltherapy or postural drainage to the patient.

15. The method of embodiment 4 comprising administering an additionalmucolytic agent selected from the group consisting of: sodium2-sulfanylethanesulfonate; disodium 2,2′-disulfanediyldiethanesulfonate;N-acetylcysteine to the patient; and combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide (SEQ ID NO. 1) and deduced amino acid (SEQID NO. 2) sequences of human DNase I as reported in Shak et al., 1990,Proc. Natl. Acad. Sci. USA 87:9188-9192. Nucleotides are numbered atleft. Amino acids are numbered above the line starting at Leu+1 of themature enzyme sequence and preceded by a 22-amino-acid putative signalsequence (underlined). The four cysteine residues are printed inboldface. Two potential N-linked glycosylation sites are indicated bylines above the amino acid sequence.

FIG. 2 shows the amino acid sequence of native human DNase I (SEQ ID NO.3) as reported in U.S. Pat. No. 6,348,343.

FIG. 3 shows the clinical timeline for the treatment of the patient inExample 1.

DETAILED DESCRIPTION

“DNA hydrolytic activity,” as used herein, refers to the enzymaticactivity of native human DNase I or a variant of human DNase I to cleaveDNA to yield 5′-phosphorylated oligonucleotide end products.

“Mucolytic agent,” as used herein, refers to an agent used to dissolvemucus in order to help loosen and clear the mucus from the airways ofthe lung.

“Patient,” as used herein, refers to a human patient.

“rhDNase I,” as used herein, refers to recombinant human DNase I, i.e.,human DNase I that is obtained by expressing a DNA construct encodinghuman DNase I in certain host cells such as Chinese hamster ovary (CHO)cells.

“A variant of human DNase I,” as used herein, refers to a polypeptidethat comprises an amino acid sequence that is different from that ofnative human DNase I but still retains at least 90% amino acid sequenceidentity with native human DNase I.

“Therapeutically effective amount,” as used herein, refers to an amountof mucolytic agent that provides a therapeutic benefit in the treatmentor management of pulmonary sarcoidosis, e.g., by delaying or minimizingone or more symptoms associated with pulmonary sarcoidosis, or byenhancing the therapeutic benefit provided by another therapeutic agentfor pulmonary sarcoidosis.

In one embodiment, the form of rhDNase I used in the methods describedherein is that found in PULMOZYME®, which contains a highly purifiedaqueous solution of rhDNase I obtained from Chinese hamster ovary (CHO)cells genetically engineered to express native human DNase I. TherhDNase I in PULMOZYME® is a glycoprotein containing 260 amino acidswith a molecular weight of 37,000 daltons. The primary amino acidsequence of this protein is identical to that of native human DNase I.Its generic name is dornase alfa. In certain embodiments, PULMOZYME® isadministered to pulmonary sarcoidosis patients in the same generalmanner (e.g., dosage, method of administration) that it is administeredto cystic fibrosis patients. In one embodiment, the form of rhDNase Iused in the methods described herein is a biosimilar of PULMOZYME®.

The DNA and amino acid sequences of native human DNase I can be found inShak et al., 1990, Proc. Natl. Acad. Sci. USA 87:9188-9192 (Shak). Alsofound in Shak is a detailed description of how a nucleotide encodinghuman DNase I may be obtained by cloning in λgt10 from a humanpancreatic cDNA library and how that nucleotide may be recombinantlyexpressed. These disclosures of Shak are incorporated by referenceherein. FIG. 1A of Shak shows the DNA and amino acid sequences of nativehuman DNase I and is reproduced herein as FIG. 1 of this application.

As an alternative to the rhDNase I found in PULMOZYME®, the methodsdescribed herein may be practiced by administering a different rhDNaseI. For example, U.S. Pat. No. 5,279,823 describes a deamidated rhDNase Ithat may be used. U.S. Pat. No. 6,348,343 describes rhDNase I variantshaving slightly different amino acid sequences from that found in nativehuman DNase I that may be used in the methods for treating pulmonarysarcoidosis described herein. For example, described are the following:

variants of human DNase I (SEQ ID NO: 3) comprising at least one aminoacid substitution at the following positions corresponding to thesequence of native human DNase I: His44, Leu45, Val48, Gly49, Leu52,Asp53, Asn56, His64, Tyr65, Val66, Val67, Ser68, Glu69, Ser94, Tyr96 orAla 114, wherein said variants have DNA hydrolytic activity;

variants of human DNase I having amino acid sequences that are at least99% identical to SEQ ID NO: 3, wherein said variants have DNA hydrolyticactivity;

variants of human DNase I having amino acid sequences that are at least95% identical to SEQ ID NO: 3, wherein said variants have DNA hydrolyticactivity;

variants of human DNase I having amino acid sequences that are at least90% identical to SEQ ID NO: 3, wherein said variants have DNA hydrolyticactivity;

variants of human DNase I having amino acid sequences that differ fromSEQ ID NO: 3 by only one amino acid substitution, wherein said variantshave DNA hydrolytic activity;

variants of a human DNase I (SEQ ID NO: 3) comprising at least one aminoacid substitution selected from the group consisting of: E13A, E13H,E13R, E13W, E13Y, H44A, H44D, H44Y, H44W, H44C, H44Q, H44N, H44E, L45C,L45K, L45R, V48C, V48K, V48R, G49C, G491, G49K, G49R, G49Y, L52C, L52K,L52M, L52N, L52R, D53A, D53K, D53R, D53Y, D53C, D53L, D53M, N56C, N56F,N56K, N56R, N56W, D58T, H64N, Y65A, Y65R, Y65W, Y65C, Y65K, Y65M, Y65S,Y65N, Y65E, Y65P, V66T, V66N, V67A, V67E, V67K, V67C, V67D, V67H, V67M,V67P, V67R, V67S, V67T, V67N, S68K, S68R, S68M, S68N, E69K, E69R, E69A,E69C, E69M, E69T, P70T, S94N, Y96T, A114C, A114E, A114G, A114H, A114K,A114L, A114M, A114Q, A114R, A114W and A114Y, where said variant has DNAhydrolytic activity;

variants of a human DNase I (SEQ ID NO: 3) comprising at least one aminoacid substitution selected from the group consisting of: E13A, E13H,E13R, E13W, E13Y, H44A, G49R, D53R, D53K, D53Y, D53A, D53C, N56R, Y65A,Y65R, Y65W, V67E, E69K, E69R A114G and A114H; and

variants of a human DNase I (SEQ ID NO: 3) comprising at least one aminoacid substitution selected from the group consisting of: H44A:D53R:Y65A,H44A:Y65A:E69R, D53R:Y65A, D53R:E69R, S94N:Y96T, V67N:E69T, Y65N:V67Tand H64N:V66T.

U.S. Pat. No. 6,391,607 also describes rhDNase I variants havingslightly different amino acid sequences from that found in native humanDNase I that may be used in the methods for treating pulmonarysarcoidosis described herein. For example, described are human DNase Ivariants comprising amino acid sequences having at least 90% identitywith the amino acid sequence of native human DNase I (SEQ ID NO: 3) anda substitution at one or more amino acid residues corresponding to Gln9,Thr14, Asn74, Ser75, and Thr205 of native human DNase I.

rhDNase I may be produced recombinantly in Chinese hamster ovary (CHO)cells by growing CHO cells that have been transfected with a suitableexpression vector encoding human DNase I in a suitable medium andpurifying the rhDNase I by conventional means, e.g., by tangential flowfiltration and column chromatography. Alternatively, rhDNase I may beproduced using other suitable recombinant host cells, as is well knownin the art.

The mucolytic agents used in the methods described herein may beadministered to the lungs by inhalation using a suitable nebulizer ornebulizer/compressor system. Suitable nebulizer/compressor systemsinclude the following:

Hudson T UP-DRAFT II® nebulizer with PULMO-AIDE® compressor;

Marquest ACORN II® nebulizer with PULMO-AIDE® compressor;

PARI LC® Jet+ nebulizer with PARI PRONEB® compressor;

PARI BABY® nebulizer with PARI PRONEB® compressor;

Durable SIDESTREAM® nebulizer with MOBILAIRE® compressor; and

Durable SIDESTREAM® nebulizer with PORTA-NEB® compressor.

When the mucolytic agent is DNase I, a dose of about 2.5 mg once dailymay be used. Alternatively, a dose of about 2.5 mg twice daily may beused. Other doses that may be used include about 0.5 mg, about 1.0 mg,about 1.5 mg, about 2.0 mg, about 2.5 mg, about 3.0 mg, about 3.5 mg,about 4.0 mg, about 4.5 mg, or about 5.0 mg, either once or twice perday. Doses of 0.5 mg to 5.0 mg, 1.0 mg to 4.0 mg, or 1.5 mg to 3.5 mg,either once or twice per day, may also be used.

In certain embodiments, the mucolytic agent is delivered by inhalationto the patient 1, 2, 3, 4, 5, 6, or 7 times per week for a certain timeperiod. In certain embodiments, the time period is about 1 week, about 2weeks, about 1 month, about 2 months, about 3 months, about 6 months,about 9 months, about 1 year, about 2 years, about 3 years, about 4years, about 5 years, about 6 years, about 7 years, about 8 years, about9 years, about 10 years, about 11 years, or about 12 years.

Pharmaceutical compositions comprising a therapeutically effectiveamount of rhDNase I and a pharmaceutically acceptable carrier orexcipient may be administered to patients in need of treatment forpulmonary sarcoidosis according to the methods described herein. Abuffered or unbuffered aqueous solution of DNase I, e.g., an isotonicsalt solution such as 150 mM sodium chloride containing 1.0 mM calciumchloride at pH 7, may be a suitable pharmaceutical composition.

In one embodiment, rhDNase I is administered as a sterile, aqueoussolution containing 1.0 mg/mL dornase alfa, 0.15 mg/mL calcium chloridedehydrate, and 8.77 mg/mL sodium chloride with no preservative. Thenominal pH of the solution is 6.3. In one embodiment, the rhDNase I issupplied in single-use ampoules that deliver 2.5 mL of this solutionthrough a nebulizer.

In certain embodiments, the methods comprise administering a mucolyticagent and an oral corticosteroid, e.g., prednisone or prednisolone, to apatient in need of treatment for pulmonary sarcoidosis. In certainembodiments, the methods comprise administering a mucolytic agent and abronchodilator to a patient in need of treatment for pulmonarysarcoidosis.

In certain embodiments, the methods comprise administering a mucolyticagent and an antibiotic to a patient with pulmonary sarcoidosis. Themucolytic agent may be DNase I and the antibiotic may be selected fromthe group consisting of TOBREX®, TOBI®, tobramycin, AKTOB®, BETHKIS®,TOBI® Podhaler, PROVENTIL®, VENTOLIN®, albuterol, ZITHROMAX®,azithromycin, Azasite, Cotazym, CREON®, ZENPEP®, Pancreaze, PERTZYE®,ULTRESA®, VIOKASE®, Nebcin, and combinations thereof. The antibiotic maybe administered by inhalation, e.g., using a nebulizer. The antibioticmay be administered together with, or separately from, the mucolyticagent.

In certain embodiments, the mucolytic agent is administered withnon-pharmaceutical therapies typically used to treat pulmonarysarcoidosis (e.g., chest physical therapy and/or postural drainage).

Example

A patient (middle aged female, African ancestry) had a history ofdebilitating chronic pulmonary sarcoidosis with multiple pulmonaryexacerbations requiring antibiotic therapy yearly. Constant coughingresulted in fractured ribs and abdominal hernias. At times, the patientwas bedridden, dyspneic, and could not climb stairs or walk more than 10feet. Steroids had been prescribed by pulmonary specialists; however theside effects of steroid therapy (diabetes, weight gain, memory loss,mental confusion, crippling joint pain) were intolerable. The patient'spulmonary sarcoidosis was deemed ultimately refractory to steroidtherapy, which was discontinued, leaving essentially no therapeuticoptions for this patient.

PULMOZYME® was prescribed to the patient by a licensed medicalpractitioner to initiate treatment. The patient responded to PULMOZYME®treatment as follows:

1. Adverse events relating to PULMOZYME®—none in more than 10 years oftreatment;

2. Coughing, wheezing, dyspnea—none (although continued PULMOZYME®therapy has been required 3-4 days/week to maintain improved pulmonaryfunction, which immediately begins to regress with periodic attempts todiscontinue therapy);

3. At present, arterial O₂ saturation is 98% on room air (finger pulseoximetry);

4. Exercise tolerance during treatment—unlimited;

5. Pulmonary exacerbations (pneumonia)—none in more than 10 years oftreatment;

6. Granuloma formation/expectoration—occasional;

7. Emergency room visits an hospitalizations relating to pulmonarysarcoidosis—none in more than 10 years of treatment;

8. Blood chemistry, liver and kidney function during treatment—normal;

9. Unrelated clinical conditions treated during PULMOZYME® therapy:

-   -   a. Abdominal hysterectomy with spinal anesthesia;    -   b. Sinusitis—two episodes treated with antihistamines;    -   c. Chest wall lipoma—surgically removed under local anesthetic;    -   d. Posterior knee lipoma—surgically removed under local        anesthetic;

10. Chest physical therapy with postural drainage—none required duringPULMOZYME® therapy;

11. Rib fractures and abdominal hernias—none during PULMOZYME® therapy;

12. General quality of life—vastly improved during PULMOZYME® therapy.

These clinical findings for treatment of chronic pulmonary sarcoidosisare even more noteworthy because black, female patients typically havemore serious pulmonary involvement with a poor long-term prognosis andvery high incidence of relapses.

The extended clinical timeline for the patient described above wasappropriate, given the nature of pulmonary sarcoidosis. Approximately50% of all pulmonary sarcoidosis patients experience remission within 2years of the onset of symptoms. It is generally accepted that Scaddingradiologic staging on presentation of the disease predicts theapproximate likelihood of remission. In some instances, remissionfollows oral steroid therapy, but in all cases the causes for remissionare unknown. Pulmonary fibrosis (Stage IV) patients do not experienceremission and only a small percentage of patients with chronic pulmonarysarcoidosis (20%±) undergo remission.

While true remission of disease in pulmonary sarcoidosis is notpredictable, end-stage disease typically progresses over a 5-10 yearperiod as the patient's condition gradually deteriorates. The typicalclinical course for chronic pulmonary sarcoidosis is a continualdeterioration of pulmonary function, culminating in acute respiratoryfailure (ARF) due variously to either: i) lung parenchymal destruction;ii) hemoptysis; iii) pneumonia; or, iv) interstitial fibrosis.

The worst clinical prognosis was associated the patient described above:

i) Age >40 at onset of disease

ii) African-American ancestry

iii) Female

iv) Requirement for steroids

v) Skin/neurologic involvement

vi) Stage II-IV chest radiograph

vii) Significant lung function impairment

viii) Recurrent pneumonias requiring ER visits, antibiotics,hospitalization, etc.

ix) Severe dyspnea with inspissated secretions on presentation

The extended clinical timeline described above allowed for a distinctionbetween remission and stabilization of the disease to be made. One wayto distinguish patients that are in remission from those that have hadtheir disease stabilized and are being maintained in a quasi-steadystate by the therapeutic intervention is to observe treatment over asuitably long interval (perhaps equivalent to the interval leading toARF and death) and periodically discontinue therapy to observe whetherthe disease symptoms worsen or remain unchanged, thereby indicating trueremission of disease. This was done on an annual basis for the patientdescribed above for the typical “survival window” (8-14 years forpatients with progressive, chronic disease).

The clinical timeline for treatment of the patient described above withPULMOZYME® is represented diagrammatically in FIG. 3 during pretreatmentand for the entire treatment interval. During treatment there has notbeen a single pulmonary exacerbation, although the patient has hadseveral surgeries and several short-duration upper respiratory tractinfections with nasal congestion, headache, and malaise. Arterialoxygenation via pulse oximetry improved from a low of SaO₂=70% duringthe pre-PULMOZYME® treatment interval to the currently measured SaO₂=99%on room air, coincident with the gradual improvement in exercise abilityand disappearance of dyspnea on exertion. The patient voluntarilydiscontinued therapy with PULMOZYME® for 2-week intervals every yearduring the treatment period to determine whether regression of diseasehas occurred or if continued therapy with PULMOZYME® is necessary. Ineach instance of discontinuation of PULMOZYME® therapy, the patient'spulmonary disease symptoms (dyspnea, inspissated secretions, etc.)worsened, but were resolved upon resumption of PULMOZYME® therapy (2.5mg qd 4× weekly). Occasionally the patient reported expectoration of“lumps” of lung tissue, presumably shed airway granulomas, and the mostrecent specimen was recovered and fixed in 10% formalin forhistopathology. The patient's routine chest x-rays have shownimprovement during treatment, with evidence of scarring that has notprogressed to more severe disease. The only temporary coughing episodesnoted during PULMOZYME® therapy have been associated with periodicbronchial irritation due to mucus plugs and/or expectoration of shedgranulomas, none of which were severe enough to result in abdominalhernia pain or rib fractures. The patient's sarcoid skin involvement isunchanged and the patient's diet has been unrestricted. The patient iscurrently in a good state of health, enjoying a markedly improvedquality of life, but must continue therapy with PULMOZYME® for theforeseeable future on the maintenance schedule of 2.5 mg qd, 4×-5×weekly.

Observations of this single patient over a long time period, with closemonitoring of the clinical signs and symptoms of this disease,demonstrated dramatic treatment-related improvements in quality of life,the complete absence of pulmonary exacerbations, and remarkableimprovement in blood oxygenation/exercise tolerance. Remission ofdisease was not evident in this patient, since annual discontinuation oftherapy with PULMOZYME® for 14 days resulted in worsening of diseasesymptoms in every instance (increased coughing, dyspnea, and physicaldiscomfort). This sequential, long-term monitoring provides a very highdegree of confidence in the beneficial results of PULMOZYME® therapy anda complete absence of side effects associated with long-term use.

Due to the progressive nature of chronic pulmonary sarcoidosis and thevery low probability of remission for this patient, it would have beendifficult to draw meaningful conclusions from observations over a shortperiod of PULMOZYME® therapy. Instead, an extended period of serialobservations was carried out, since only a single patient was studiedand there was no test or indicator that could be used to evaluate theprogression or remission status of the disease in a single patient—otherthan discontinuation of therapy.

Six fundamentally important questions were affirmatively addressed inthe treatment described above:

1. Does the short treatment interval (1 yr) predict the long termresponse (10 yrs) when lifetime therapy is anticipated (Kravitz et al.,2009, Contemp. Clin. Trials 30(5):436-445)?

2. Can the therapeutic intervention be tolerated indefinitely without anadverse drug response (ADR)?

3. Will the experimental design be able to separate spontaneousremission of disease from the continuing need for maintenance therapy ina steady-state patient disequilibrium?

4. Is the improvement in patient quality of life justified by the costand effort required by therapy and what are the assessment tools neededto evaluate long-term treatment?

5. Can the n=1 trial be continued until treatment efficacy andtolerability is either established or disproved (Guyatt et al., 1986, N.Engl. J. Med. 314(14):889-892; Rochon, 1990, J. Clin. Epidemiol.43(5):499-508)?

6. Can the n=1 results be used to develop stratified treatment arms forsubsequent larger population based RCT's that would be essential forformal marketing approval of the therapeutic intervention by the US FDA?

In answering the above questions, the objective was to utilize this n=1stratified medicine approach for the index chronic pulmonary sarcoidosispatient to support initiatives that facilitate “evidence-based” medicine(Guyatt et al., 2000, Evidence-Based Medicine Working Group, JAMA284(10):1290-1296; Sackett et al., 1996, BMJ 312(7023):71-72; Lauer &Collins, 2010, JAMA 303(21):2182-2183; Collins, 2010, Science327(5961):36-37) and, more importantly, quickly maximize therapeuticprecision for a patient in severe distress with no alternative treatmentoptions. The overarching goal was to determine the optimalindividualized treatment using objective, results-driven criteriaevolved over the course of therapy. Based on the objective results ofthis n=1 trial, this patient has helped to provide key insights intoidentifying how individual outcomes might be improved within the largerheterogeneous at-risk population by establishing individual treatmentstratifications based on specific clinical risk profiles and the stageand spectrum of disease. Furthermore, as a part of the individualizedtherapy, annual no-treatment (“washout”) intervals with the indexpatient were utilized over time to periodically evaluate the continuedbenefits of long-term therapy.

One downside to the n=1 clinical trial is the constant, long-termmonitoring required and associated patient recruitment and retentionissues. For the index patient in this application, recruitment,retention and compliance were not a problem since no alternative therapyexisted and the patient's distressed condition at the outset gave riseto a discouraging, steadily declining medical prognosis coupled with anunwillingness on the part of the patient to undergo long periods ofsuboptimal treatment with previously attempted ineffective standard care(see the Clinical Timeline shown in FIG. 3).

A second downside to the n=1 clinical trial relates to the confoundingeffects of lifestyle changes (dietary modification, other healthcareissues, exercise regimens, etc.) on interpretation of treatment results.But these confounding variables were essentially averaged out over timefor the index patient. It is here that increasing the length of thetrial (see Clinical Timeline) clearly resolved ambiguities relating toconfounding variable effects, including the possibility of spontaneousremission of disease. Washout periods (annual 2-week “no-treatment”intervals) were invariably associated with return of disease symptoms,indicating that the index chronic pulmonary sarcoidosis patient may beanalogous to the CF patient requiring quasi-daily therapy; furthermore,the patient's subjective experience confirmed the utility of treatmentsand adverse drug responses (ADRs) were not observed over the entiretreatment interval reported herein.

PULMOZYME® is clearly validated as a safe and effective life-longtreatment regimen based upon the study length and the index patient'ssubjective and objective responses. It should be noted that the patientwas monitored daily during washout intervals to avoid compromisingpatient safety—an approach analogous to the placebo arm of a RCT. Inaddition, the patient was instructed to immediately resume treatment ifher physical condition (dyspnea, excessive coughing, discolored mucus,etc.) deteriorated. Typically, these disease symptoms began to recurbetween week 1 and week 2, prior to the end of the 2 week washoutinterval.

From the long-term results of this index patient, it is evident thatcertain stratifications of future patient groups in a standard RCT wouldbe appropriate, although such efforts would likely cost tens of millionsof dollars, involve multiple international clinical centers, and areasonably large clinical research team. The work presented herein iskey to establishing the next phase of clinical studies, raisingsufficient funding and completing RCTs that would hopefully lead to USFDA approval of PULMOZYME® for use in chronic pulmonary sarcoidosis.Recognizing that the USA is in the grip of a healthcare crisis hasmotivated serious calls for advances in medical research (Collins, 2010,Science 327(5961):36-37) and adoption of precision medicine approachesfor rare diseases, of which chronic pulmonary sarcoidosis is one thathas stubbornly defied successful treatment until now.

Long-term PULMOZYME® therapy of the above patient has been demonstratedto dramatically reduce the morbidity of chronic pulmonary sarcoidosisand to improve quality of life. Disease remission does not appear tohave occurred during the treatment interval for this patient andclinical improvement was entirely dependent upon continued PULMOZYME®therapy. While symptoms of pulmonary sarcoidosis have been and remainmitigated for this patient, the underlying cause of the disease remainsand there is potential for rapid disease progression upondiscontinuation of therapy.

What is claimed is:
 1. A method of treating pulmonary sarcoidosiscomprising administering to a patient in need thereof a therapeuticallyeffective amount of a mucolytic agent.
 2. The method of claim 1 wherethe mucolytic agent is DNase I.
 3. The method of claim 1 where themucolytic agent is selected from the group consisting of: sodium2-sulfanylethanesulfonate, disodium 2,2′-disulfanediyldiethanesulfonate,and combinations thereof.
 4. The method of claim 1 where the DNase I isrecombinant DNase I.
 5. The method of claim 4 where the recombinantDNase I is recombinant human DNase I.
 6. The method of claim 5 where therecombinant human DNase I is administering to the patient's lungs byinhalation.
 7. The method of claim 6 where the inhalation carried outwith the use of a nebulizer.
 8. The method of claim 5 where therecombinant human DNase I has the amino acid sequence of native humanDNase I.
 9. The method of claim 5 where the recombinant human DNase Ihas the amino acid sequence of SEQ ID NO.
 3. 10. The method of claim 1where the pulmonary sarcoidosis is acute pulmonary sarcoidosis.
 11. Themethod of claim 1 where the pulmonary sarcoidosis is chronic pulmonarysarcoidosis.
 12. The method of claim 1 comprising administering anantibiotic to the patient.
 13. The method of claim 1 comprisingadministering a bronchodilator to the patient.
 14. The method of claim 1comprising administering chest physical therapy or postural drainage tothe patient.
 15. The method of claim 4 comprising administering anadditional mucolytic agent selected from the group consisting of: sodium2-sulfanylethanesulfonate; disodium 2,2′-disulfanediyldiethanesulfonate;N-acetylcysteine to the patient; and combinations thereof.